CN111735950B - FGF18 and CA125 combined used as early ovarian cancer biomarker and kit - Google Patents
FGF18 and CA125 combined used as early ovarian cancer biomarker and kit Download PDFInfo
- Publication number
- CN111735950B CN111735950B CN202010694119.9A CN202010694119A CN111735950B CN 111735950 B CN111735950 B CN 111735950B CN 202010694119 A CN202010694119 A CN 202010694119A CN 111735950 B CN111735950 B CN 111735950B
- Authority
- CN
- China
- Prior art keywords
- fgf18
- ovarian cancer
- antibody
- kit
- stage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4725—Mucins, e.g. human intestinal mucin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/50—Fibroblast growth factors [FGF]
Abstract
The invention relates to a combination of FGF18 and CA125 as early ovarian cancer biomarkers and a kit, belonging to the technical field of immunodetection. The present invention discloses the use of an agent for detecting FGF18 and CA125 levels in a sample from a subject for the preparation of a kit for predicting, evaluating or diagnosing early ovarian cancer in said subject. The invention also provides a kit for predicting, evaluating or diagnosing the early ovarian cancer of a subject, which can be effectively used for early screening of the ovarian cancer, and the sensitivity and specificity of the prediction, evaluation and diagnosis of the ovarian cancer in the subject are obviously improved and the false positive rate in early ovarian cancer detection is greatly reduced by the kit provided by the invention.
Description
Technical Field
The present invention relates generally to the field of immunodetection technology, and in particular to the use of FGF18 and CA125 in combination as early ovarian cancer biomarkers, and kits made therefrom.
Background
Ovarian cancer is one of the common malignant tumors of female reproductive organs, and causes serious threat to female life. The ovarian is deep in the pelvic cavity, small in size and lack of typical symptoms during onset, so that early diagnosis of ovarian cancer is difficult, and the early diagnosis becomes a technical problem. Clinical findings have often progressed to mid-to late-stages. And to middle and late stages, particularly after metastasis, even when treated by ovarian epithelial cancer surgery, the recurrence rate and 5-year survival rate are low. Thus, early diagnosis of ovarian cancer is important for reducing metastasis and improving 5-year survival rate. At present, the diagnosis of ovarian cancer is clinically based on vaginal ultrasonic examination (TVU) and detection of ovarian cancer markers in blood
CA-125 (carbohydrate antigen (carbohydrate antigen) 125), also known as mucin-16, is a protein encoded by the MUC16 gene. CA125 is one of the markers of ovarian epithelial cancer and is widely used for diagnosis and follow-up of ovarian epithelial cancer at present. However, CA125 is also increased to a varying extent in the case of certain non-malignant diseases, and thus, the specificity and sensitivity are not ideal when CA125 is used to diagnose ovarian cancer. In recent years, the use of a combined detection of epididymal protein 4 (epididymis protein 4) and CA125 for screening and diagnosis of ovarian cancer has been proposed (patent documents 1 and 2). However, single HE4 has a higher sensitivity to stage I ovarian cancer assisted diagnosis than CA125, but also only 45.9%; the sensitivity of the combined HE4 and CA125 detection was not improved in stage I ovarian cancer (see Moore RG, brown AK, miller MC et al, the use of multiple novel tumor biomarkers for the detection of ovarian carcinoma in patients with a pelvic mass. Gynecol Oncol,2008, 108:402-408). Therefore, there is an urgent need to develop new diagnostic early ovarian cancer serum markers in the hope of being able to sensitively and specifically determine the occurrence of ovarian cancer through peripheral blood detection at the early stages of ovarian cancer.
Human fibroblast growth factor 18 (Fibroblast Growth Factor, FGF 18) is encoded in humans by the FGF18 gene. FGF18 is a member of the Fibroblast Growth Factor (FGF) family. FGF family members have a broad range of mitogenic and cell survival activities and are involved in a variety of biological processes including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion. However, there has never been disclosed a biomarker for the combined use of CA125 and FGF18 for early diagnosis of ovarian cancer.
[ reference ] to
[ patent document 1]: CN108008132a;
[ patent document 2]: CN103954761a.
Disclosure of Invention
The present invention relates generally to early ovarian cancer biomarkers and in particular to methods and uses for predicting, evaluating and diagnosing early ovarian cancer by measuring the level of the biomarker combination FGF18 and CA125, and also provides kits for predicting, evaluating and diagnosing early ovarian cancer. The methods and kits provided herein are particularly useful for epithelial ovarian cancer, and more particularly for early stage ovarian cancer (i.e., stage I or stage II ovarian cancer), thereby providing a method and kit that is easy to handle, highly sensitive, highly specific, and can be effectively used for early screening of ovarian cancer. In order to achieve the above object of the present invention, the present invention adopts the following technical scheme:
in one aspect, the invention provides the use of an agent that detects FGF18 and CA125 levels in a sample from a subject in the preparation of a kit for predicting, evaluating or diagnosing early stage ovarian cancer in the subject. In some embodiments, the ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer, or both stage I and stage II ovarian cancer.
In some embodiments, the agent that detects FGF18 and CA125 levels is an antibody to FGF18 and an antibody to CA125, respectively. Preferably, the antibody against FGF18 and the antibody against CA125 are a monoclonal antibody against FGF18 and a monoclonal antibody against CA125, respectively.
In some embodiments, the sample is a biological fluid sample from the subject. Preferably, the sample is blood, serum, plasma, lymph, cerebrospinal fluid, ascites, urine and tissue biopsies from the subject. More preferably, the sample is blood, serum, and plasma from the subject.
In some embodiments, the kit is a chemiluminescent kit. Preferably, the chemiluminescent kit comprises a capture antibody, a detection antibody, and a chemiluminescent substrate.
In some embodiments, the capture antibody is selected from the group consisting of FGF18 monoclonal antibody linked to a magnetic bead, CA125 monoclonal antibody linked to a magnetic bead, and combinations thereof. Alternatively, the detection antibody is selected from the group consisting of FGF18 monoclonal antibodies with a label of alkaline phosphatase, CA125 monoclonal antibodies with a label of alkaline phosphatase, and combinations thereof. Alternatively, the chemiluminescent substrate is AMPPD (CAS No. 122341-56-4).
In another aspect, the invention provides a kit for predicting, evaluating or diagnosing ovarian cancer in a subject comprising reagents for detecting FGF18 and CA125 levels in a sample.
Preferably, the ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer, or both stage I and stage II ovarian cancer.
In some embodiments, the agent that detects FGF18 and CA125 levels is an antibody to FGF18 and an antibody to CA125, respectively. Preferably, the antibody against FGF18 and the antibody against CA125 are a monoclonal antibody against FGF18 and a monoclonal antibody against CA125, respectively.
In some embodiments, the sample is a biological fluid sample from the subject. Preferably, the sample is blood, serum, plasma, lymph, cerebrospinal fluid, ascites, urine and tissue biopsies from the subject. More preferably, the sample is blood, serum, and plasma from the subject.
In some embodiments, the kit is a chemiluminescent kit. In some embodiments, the kit is a diagnostic kit. Preferably, the chemiluminescent kit comprises a capture antibody, a detection antibody, and a chemiluminescent substrate.
In some embodiments, the capture antibody is selected from the group consisting of FGF18 monoclonal antibody linked to a magnetic bead, CA125 monoclonal antibody linked to a magnetic bead, and combinations thereof. Alternatively, the detection antibody is selected from the group consisting of FGF18 monoclonal antibodies with a label of alkaline phosphatase, CA125 monoclonal antibodies with a label of alkaline phosphatase, and combinations thereof. Alternatively, the chemiluminescent substrate is AMPPD.
In some embodiments, the kit comprises 2 reagent subgroups, wherein subgroup 1 comprises: magnetic beads coated with a monoclonal antibody directed against FGF18, a FGF18 monoclonal antibody having an alkaline phosphatase label, and a chemiluminescent substrate AMPPD; and is also provided with
Wherein subgroup 2 comprises: magnetic beads coated with a monoclonal antibody directed against CA125, a CA125 monoclonal antibody with an alkaline phosphatase label, and a chemiluminescent substrate AMPPD.
In another aspect of the present application, there is also provided a method of predicting, evaluating or diagnosing the presence of ovarian cancer and stage of development thereof in a subject, comprising detecting FGF18 and CA125 levels in a sample from the subject. In some embodiments, the level of FGF18 and CA125 in a sample of a subject can be detected by a kit as provided in the above aspects. Alternatively, the detected levels of FGF18 and CA125 in a sample of a subject can be compared to FGF18 and CA125 level values (i.e., standard values) obtained from a non-diseased population to determine whether ovarian cancer is present and its stage of development in the subject.
Advantageous effects
The present invention uses FGF18 in combination with CA125 as a novel marker for ovarian cancer screening and diagnosis. The inventors have demonstrated that there is a significant difference in serum FGF18 and CA125 levels between the ovarian cancer patient group and the healthy control group (P < 0.01), and that there is a correlation between serum FGF18 and CA125 levels in the ovarian cancer patient group (r=0.812). Compared with the single detection of one of the markers, the sensitivity and the specificity of the ovarian cancer diagnosis method and the kit provided by the invention are both obviously improved (the sensitivity is 86.9 percent and the specificity is 92.1 percent), the false positive rate is greatly reduced, and the method for early ovarian cancer screening with low misdiagnosis rate, reduced human error and improved accuracy and the corresponding chemiluminescent detection kit are provided.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention clearer, the present invention provides the following drawings:
FIG. 1 shows a standard curve for detecting FGF18 protein by chemiluminescence.
FIG. 2 shows a standard curve for detecting CA125 protein by chemiluminescence.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Some terms related to the present invention are explained as follows.
The biomarker "CA-125", saccharide antigen (carbohydrate antigen) 125, also known as mucin-16, is a protein encoded by the MUC16 gene. CA125 is one of the markers of ovarian epithelial cancer and is widely used for diagnosis and follow-up of ovarian epithelial cancer at present. Herein, "CA-125" means a polypeptide biomarker, or fragment thereof, having at least 85% sequence identity to NCBI accession number NP-078966.2.
HUMAN fibroblast growth factor 18 (Fibroblast Growth Factor, FGF 18) has an accession number uniprotKB-O76093 (FGF18_HUMAN) in uniprotrot.org. FGF18 proteins are encoded in humans by the FGF18 gene, 207 amino acids in length, which is a member of the Fibroblast Growth Factor (FGF) family, which has a wide range of mitogenic and cell survival activities and is involved in a variety of biological processes including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion. The protein was shown to induce neurite outgrowth in PC12 cells in vitro. Studies of similar proteins in mice and chickens have shown that the protein is a pleiotropic growth factor that stimulates proliferation in a variety of tissues, especially the liver and small intestine. Knockout studies of similar genes in mice indicate that this protein plays a role in regulating proliferation and differentiation of midline cerebellum structures. Herein, "FGF18" means a polypeptide biomarker or fragment thereof having at least 85% sequence identity to NCBI accession No. np_ 003853. Specifically, the full-length amino acid sequence of FGF18 according to accession number np_003853 is shown below:
MYSAPSACTCLCLHFLLLCFQVQVLVAEENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARGEDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKECVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHFMKRYPKGQPELQKPFKYTTVTKRSRRIRPTHPA。
in this context, the types of ovarian cancer predicted, assessed or diagnosed are serous, endometrioid, mucinous and clear cell tumors. Also, predicting, evaluating or diagnosing ovarian cancer includes predicting a particular stage of a disease, such as stage I (IA, IB or IC), stage II, stage III and stage IV tumors. Additionally, "early stage of ovarian cancer" or "early stage ovarian cancer" means ovarian cancer in stage I or stage II. By "diagnosis" is meant identifying the presence or nature of a disorder (i.e., ovarian cancer). Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of individuals with positive disease in a population that are tested to be total individuals with disease. The "specificity" of a diagnostic assay is 1 minus the false positive rate, where "false positive" rate refers to the proportion of individuals tested positive but not actually diseased.
By "chemiluminescent kit" is meant a kit for determining the level of a biomarker using a chemiluminescent enzyme immunoassay (Chemiluminescent enzyme immunoassay, cLEIA). Specifically, in chemiluminescent enzyme immunoassay, an enzyme is used for labeling a bioactive substance for immune reaction, enzyme on an immune reaction complex acts on a luminescent substrate again, luminescence is generated under the action of a signal reagent, and then luminescence measurement is performed by a luminescence signal measuring instrument, so that the biomarker level in a sample from a subject is quantitatively analyzed.
In some embodiments of the present application, the kits of the present application include a capture antibody, a detection antibody, and a chemiluminescent substrate. Specifically, the capture antibodies can be prepared by linking a monoclonal antibody directed against the biomarker of interest to magnetic beads in the presence of a coupling solution under appropriate reaction conditions. Advantageously, the capture antibody can be obtained by preparing magnetic beads coated with murine anti-human FGF18 monoclonal antibody by coating the carboxylic magnetic beads with a suitable concentration of murine anti-human FGF18 monoclonal antibody in the presence of a coupling solution, at 37 ℃ for 2 hours, or at 4 ℃ overnight.
Further, the detection antibody may be an FGF18 monoclonal antibody having a basic phosphatase label, a CA125 monoclonal antibody having a basic phosphatase label, and combinations thereof. Alternatively, the chemiluminescent substrate is AMPPD.
It is noted that while the level of a biomarker in a sample may be detected by other methods known in the art, such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence chromatography, electrochemiluminescence, etc., the inventors have unexpectedly found that when combining a chemiluminescent method with the biomarkers CA125 and FGF18 of the present application, at least the following advantages can be achieved:
1. early stage ovarian cancer patients are detected with high sensitivity (e.g., 10-22 mol/L). This is far above the detection limit of other immunoassay methods such as radioimmunoassays and enzyme-linked immunoassays.
2. Has a wide linear power range. In the methods and kits of the present application, the luminous intensity is linear between 4-6 orders of magnitude and the concentration of the assay substance. This range is far greater than the linear range (2.0) of absorbance (OD value) in the enzyme immunoassay.
3. The result is stable, the error is small, the sample directly emits light, no light source is needed for irradiation, and the influence of a plurality of possible factors (light source stability, light scattering, light wave selector and the like) on analysis is eliminated, so that the analysis is sensitive, stable and reliable.
Thus, the combination of chemiluminescence with the biomarkers CA125 and FGF18 of the present application has the advantages described above.
Examples
The product purchase information used in the examples is as follows, but is not limited to the same manufacturer.
Murine anti-human FGF18 monoclonal antibody (coated antibody): cargo number: sc-393471, available from Santacruz under the trade name FGF-18 antibody.
Murine anti-human FGF18 monoclonal antibody (detection antibody): cargo number: ab86571 is available from Abcam under the trade name Anti-FGF18 antibody.
FGF18 protein standard: cargo number: 13206-H08H, available from SinoBiological under the trade name FGF18 protein.
Murine anti-human CA125 monoclonal antibody (coated antibody), cat: CA125-McAb2 is purchased from Phpeng organisms under the trade name carbohydrate antigen 125 antibody.
Murine anti-human CA125 monoclonal antibody (detection antibody), cat No.: CA125-McAb1 is purchased from Phpeng organism under the trade name carbohydrate antigen 125 antibody.
CA125 protein standard: cargo number: CA125-Ag is purchased from the Phpeng organism under the trade name carbohydrate antigen 125.
Magnetic beads: cargo number: magCOOH, available from SUZHIYI microsphere technology Co., ltd, under the trade name of carboxyl magnetic beads.
Alkaline phosphatase: cargo number: SP011401, commercially available from chinese medicine under the trade name alkaline phosphatase.
AMPPD: CAS No. 122341-56-4, 4-methoxy-4- (3-phosphorylphenyl) spiro [1, 2-dioxycyclohexane-3, 2' -adamantane ], is an alkaline phosphatase substrate available from Beijing Gekko as a chemiluminescent substrate.
Tween-20: purchased from an organism under the trade name tween-20.
Example 1. Establishment of FGF18 assay System and optimization thereof
And (3) constructing a detection system: coating carboxyl magnetic beads with 2.5 mug/mL of the mouse anti-human FGF18 monoclonal antibody at 37 ℃ for 2 hours or coating magnetic beads at 4 ℃ overnight, so as to prepare the magnetic beads coated with the mouse anti-human FGF18 monoclonal antibody, namely a capture antibody; then, FGF18 protein standard substances and serum samples with the concentration of 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 0ng/ml are respectively added into different reaction cups to react for 30 minutes at 37 ℃; then adding alkaline phosphatase-labeled murine anti-human FGF18 monoclonal antibody (i.e., detection antibody) at a concentration of 0.25 μg/mL, reacting at 37℃for 30 minutes, washing the magnetic beads, and removing the supernatant; finally, a luminescent substrate AMPPD is added, and the luminescence value is measured. According to the luminescence values of the reaction system obtained from standard product values of different concentrations, FGF18 protein standard curve is constructed, and the result is shown in FIG. 1. As can be seen from FIG. 1, the linear range of the FGF18 detection system is 0ng/mL-1000ng/mL, the linear correlation coefficient r of the standard substance in the linear range is more than or equal to 0.990, and the recovery rate is 90-110%.
And (3) determining a detection system: antibodies of different concentrations were detected by checkerboard Fang Zhenfa, wherein the capture antibodies were selected from 0.5, 1.5, 2.5, 3.5, 4.5, 5.5 μg/mL, and the detection antibodies were selected from 0.05, 0.15, 0.2, 0.25, 0.35, 0.45, 0.55 μg/mL. The antibodies with different concentrations are detected, and the optimal working concentration of the FGF18 capture antibody is found to be 2.5 mug/mL, and the optimal working concentration of the FGF18 detection antibody is found to be 0.25 mug/mL.
EXAMPLE 2 FGF18 chemiluminescent assay kit
The FGF18 chemiluminescence detection kit is constructed according to the FGF18 serum detection system established in the example 1, and the specific components are shown in the table 1:
TABLE 1 FGF18 chemiluminescent detection kit components
Evaluating FGF18 chemiluminescent assay kit: detecting the FGF18 positive quality control product by using an FGF18 chemiluminescence detection kit, wherein the detection is repeated for 10 times at two levels of 62.5ng/mL and 500ng/mL of FGF18 protein concentration respectively, and the result shows that the variation coefficient CV is less than or equal to 10%; when the same sample is detected by 3 lot number kits, the variation coefficient CV between lots of the 3 lot number kits is less than or equal to 12%.
Example 3 establishment of a CA125 serum detection reaction System and optimization thereof
Coating magnetic beads with a murine anti-human CA125 monoclonal antibody at a concentration of 5 μg/mL at 37℃for 2 hours or at 4℃overnight; adding the CA125 protein standard substance and the serum sample with the concentration of 0U/mL,10U/mL,20U/mL,40U/mL,80U/mL,160U/mL and 320U/mL into a reaction hole respectively, and reacting for 30 minutes at 37 ℃; then, the reaction is carried out for 30 minutes at 37 ℃ by using a mouse anti-human CA125 monoclonal antibody marked by alkaline phosphatase with the concentration of 0.5 mug/mL, and the magnetic beads are washed to remove the supernatant; finally, a luminescent substrate AMPPD is added, and the luminescence value is measured. A standard curve of CA125 was constructed based on luminescence values of the reaction system obtained from standard product values of different concentrations, and the results are shown in FIG. 2. As shown in FIG. 2, the linear range of the CA125 chemiluminescent detection kit is 10U/mL-320U/mL, the linear correlation coefficient r of the standard substance in the linear range is more than or equal to 0.990, and the recovery rate is in the range of 90-110%.
And (3) determining a detection system: similar to the checkerboard method of example 2, the best working concentration of the self-made CA125 capture antibody was found to be 5 μg/mL and the best working concentration of the CA125 detection antibody was found to be 0.5 μg/mL by detecting antibodies at different concentrations.
The main components of the detection system were determined by the above study, and then a CA125 serum detection system was established.
Example 4 CA125 chemiluminescent detection kit
The CA125 chemiluminescent detection kit is constructed according to the CA125 serum detection system established in example 3, and the specific components are shown in the following table 2:
TABLE 2 CA125 chemiluminescent detection kit components
Evaluation of CA125 chemiluminescent detection kit: detecting a CA125 repeatability reference by using a CA125 chemiluminescence detection kit, wherein the repeated detection is carried out for 10 times at two levels of the CA125 protein concentration of 20U/mL and 80pg/mL respectively, and the result shows that the variation coefficient CV is less than or equal to 10%; when the same sample is detected by 3 lot number kits, the variation coefficient CV between lots of the 3 lot number kits is less than or equal to 15%.
Example 5 Combined detection kit for human ovarian cancer marker FGF18-CA125
The FGF18 chemiluminescence detection kit constructed in the example 2 and the CA125 chemiluminescence detection kit constructed in the example 4 are combined to form an FGF18-CA125 combined detection kit.
Example 6 diagnosis and prognosis of early ovarian cancer with FGF18-CA125 Joint detection kit
100 clinical samples were collected, 22 samples of the normal population, 78 samples of early stage ovarian cancer patients, and 1mL of serum per sample. Of the 78 ovarian cancer patients, 24 were stage I ovarian cancer patients and 54 were stage II ovarian cancer patients. Detecting the concentration of FGF18 and CA125 markers in serum of an ovarian cancer patient and healthy normal person respectively, analyzing the obtained data by utilizing SPASS statistical software according to the detection result, and if the difference between different groups has statistical significance, performing independent sample t-test, wherein the result is expressed as x+/-s; correlation analysis was performed on FGF18 and CA125 for each group, with P <0.05 as the difference, statistically significant. Finally, the specificity and sensitivity of FGF18 and CA125 markers were tested individually or in combination according to data statistics, and the results are shown in table 3 for ovarian cancer group serum FGF18 (251± 114.51) ng/mL; CA125 (591.96 + -384.5) U/mL, both higher than FGF18 (132+ -83.82) ng/mL of healthy control group; CA125 (30.45+ -25.87) U/mL was statistically processed, and the difference was statistically significant (p < 0.01).
TABLE 3 serum FGF18 and CA125 test results (X.+ -.S) for ovarian cancer and healthy control
| Group of | n | Serum FGF18 (ng/mL) | CA125(U/mL) |
| Healthy control group | 22 | 132±83.82 | 30.45±25.87 |
| Ovarian cancer | 78 | 251±114.51 | 591.96±384.5 |
Note that: the ovarian cancer group is compared with the healthy control group, and P is less than 0.05; comparison of serum FGF18 between ovarian cancer group and healthy control group, P < 0.05; comparison of serum CA125 levels between ovarian cancer group and healthy control group, P < 0.05.
Meanwhile, there was a correlation between the levels of FGF18 and CA125 in the ovarian cancer group (r=0.812), whereas there was no significant correlation between the levels of FGF18 and CA125 in the healthy control group (r=0.017), and the results are shown in table 4.
TABLE 4 correlation comparison between FGF18 and CA125 for ovarian cancer groups and healthy control groups
| Group of | n | Serum FGF18 (ng/mL) | CA125(U/mL) | Correlation coefficient r |
| Healthy control group | 22 | 132±83.82 | 30.45±25.87 | 0.017 |
| Ovarian cancer | 78 | 251±114.51 | 591.96±384.5 | 0.812 |
Note that: there was a significant correlation between the ovarian cancer group CA125 and FGF18 levels (r=0.812), whereas there was no significant correlation between the healthy control group CA125 and FGF18 levels (r=0.017).
Further, by sensitivity and specificity analysis of ovarian cancer diagnosis cases, it is known that: the sensitivity of detecting FGF18 marker alone is 72.3% and the specificity is 85.4%; the sensitivity of the CA125 marker alone was 82.5% and the specificity was 70.1%. The sensitivity was 86.9% and the specificity was 92.1% when FGF18 and CA125 markers were combined (see table 5 below). From this, the combined detection of the two markers FGF18 and CA125 was significantly better than the single ovarian cancer marker detection.
Table 5. Results of diagnosis of ovarian cancer by using FGF18-CA125 combined detection kit
| Marker(s) | Area under curve | Sensitivity of | Specificity (specificity) |
| FGF18 | 0.792 | 72.3％ | 85.4％ |
| CA125 | 0.834 | 82.5％ | 70.1％ |
| FGF18+CA125 | 0.892 | 86.9％ | 92.1％ |
Note that: the combined detection of the FGF18 and the CA125 markers is significantly superior to the detection of a single ovarian cancer marker in terms of sensitivity and specificity.
In conclusion, FGF18 and CA125 can be used as markers for diagnosing and predicting early ovarian cancer, can be used for early diagnosis of ovarian cancer, and improves accuracy of early prediction.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (8)
1. Use of an agent that detects FGF18 and CA125 levels in a sample from a subject, wherein the ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer, or both stage I and stage II ovarian cancer, the sample being blood, serum, plasma from the subject, in the manufacture of a kit for predicting, evaluating, or diagnosing ovarian cancer in the subject; the reagents for detecting FGF18 and CA125 levels are an antibody to FGF18 and an antibody to CA125, respectively; the optimal working concentration of the FGF18 antibody is 0.25 mug/mL, and the optimal working concentration of the CA125 antibody is 0.5 mug/mL; the antibody is a monoclonal antibody.
2. The use according to claim 1, wherein the kit is a chemiluminescent kit comprising a capture antibody, a detection antibody and a chemiluminescent substrate.
3. The use according to claim 2, wherein the capture antibody is selected from the group consisting of FGF18 monoclonal antibody linked to magnetic beads, CA125 monoclonal antibody linked to magnetic beads, and combinations thereof;
the detection antibody is selected from the group consisting of FGF18 monoclonal antibodies with alkaline phosphatase labels, CA125 monoclonal antibodies with alkaline phosphatase labels, and combinations thereof; and/or the number of the groups of groups,
the chemiluminescent substrate is AMPPD.
4. A kit for predicting, evaluating or diagnosing ovarian cancer in a subject comprising reagents for detecting the levels of FGF18 and CA125 in a sample,
wherein the ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer, or both stage I and stage II ovarian cancer, and the sample is blood, serum, plasma from the subject.
5. The kit of claim 4, wherein the reagents for detecting FGF18 and CA125 levels are an antibody to FGF18 and an antibody to CA125, respectively.
6. The kit of claim 5, wherein the antibody to FGF18 and the antibody to CA125 are a monoclonal antibody to FGF18 and a monoclonal antibody to CA125, respectively.
7. The kit of claim 4, wherein the kit is a chemiluminescent kit comprising a capture antibody, a detection antibody, and a chemiluminescent substrate, wherein: the capture antibody is selected from the group consisting of FGF18 monoclonal antibody linked to a magnetic bead, CA125 monoclonal antibody linked to a magnetic bead, and combinations thereof;
the detection antibody is selected from the group consisting of FGF18 monoclonal antibodies with alkaline phosphatase labels, CA125 monoclonal antibodies with alkaline phosphatase labels, and combinations thereof; and/or the number of the groups of groups,
the chemiluminescent substrate is AMPPD.
8. The kit of claim 6, wherein the kit comprises 2 reagent subgroups,
wherein subgroup i comprises: magnetic beads coated with a monoclonal antibody directed against FGF18, a FGF18 monoclonal antibody having an alkaline phosphatase label, and a chemiluminescent substrate AMPPD; and is also provided with
Wherein subgroup II comprises: magnetic beads coated with a monoclonal antibody directed against CA125, a CA125 monoclonal antibody with an alkaline phosphatase label, and a chemiluminescent substrate AMPPD.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010694119.9A CN111735950B (en) | 2020-07-17 | 2020-07-17 | FGF18 and CA125 combined used as early ovarian cancer biomarker and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010694119.9A CN111735950B (en) | 2020-07-17 | 2020-07-17 | FGF18 and CA125 combined used as early ovarian cancer biomarker and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111735950A CN111735950A (en) | 2020-10-02 |
| CN111735950B true CN111735950B (en) | 2023-07-21 |
Family
ID=72655970
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010694119.9A Active CN111735950B (en) | 2020-07-17 | 2020-07-17 | FGF18 and CA125 combined used as early ovarian cancer biomarker and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111735950B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112684175B (en) * | 2021-01-23 | 2021-09-24 | 上海科语生物科技有限公司 | Kit for detecting ovarian cancer |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006135886A2 (en) * | 2005-06-13 | 2006-12-21 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| WO2009015050A2 (en) * | 2007-07-20 | 2009-01-29 | The Gov. Of The U.S.A. As Represented By The Secretary Of The Department Of Health & Human Services | Gene expression profile for predicting ovarian cancer patient survival |
| CN101680038A (en) * | 2007-04-13 | 2010-03-24 | 百时美施贵宝公司 | Biomarkers and methods for determining sensitivity to vascular endothelial growth factor receptor-2 modulators |
| GB201607393D0 (en) * | 2016-04-28 | 2016-06-15 | Isis Innovation | Biomarkers for early diagnosis of ovarian cancer |
| CN107823630A (en) * | 2011-11-14 | 2018-03-23 | 戊瑞治疗有限公司 | The method for the treatment of cancer |
| WO2019232485A1 (en) * | 2018-05-31 | 2019-12-05 | Nvigen, Inc. | Accurate blood test to predict cancer incidence, recurrence, guide and monitor treatment intervention |
-
2020
- 2020-07-17 CN CN202010694119.9A patent/CN111735950B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006135886A2 (en) * | 2005-06-13 | 2006-12-21 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| CN101680038A (en) * | 2007-04-13 | 2010-03-24 | 百时美施贵宝公司 | Biomarkers and methods for determining sensitivity to vascular endothelial growth factor receptor-2 modulators |
| WO2009015050A2 (en) * | 2007-07-20 | 2009-01-29 | The Gov. Of The U.S.A. As Represented By The Secretary Of The Department Of Health & Human Services | Gene expression profile for predicting ovarian cancer patient survival |
| CN107823630A (en) * | 2011-11-14 | 2018-03-23 | 戊瑞治疗有限公司 | The method for the treatment of cancer |
| GB201607393D0 (en) * | 2016-04-28 | 2016-06-15 | Isis Innovation | Biomarkers for early diagnosis of ovarian cancer |
| WO2019232485A1 (en) * | 2018-05-31 | 2019-12-05 | Nvigen, Inc. | Accurate blood test to predict cancer incidence, recurrence, guide and monitor treatment intervention |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111735950A (en) | 2020-10-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Haab | Methods and applications of antibody microarrays in cancer research | |
| CN111521807B (en) | Spondin1 and CA125 combined used as early ovarian cancer biomarker and kit | |
| Huang et al. | A biotin label-based antibody array for high-content profiling of protein expression | |
| US8541183B2 (en) | Methods of identification, assessment, prevention and therapy of lung diseases and kits thereof | |
| US20180238890A1 (en) | Methods and materials for detection, diagnosis and management of ovarian cancer | |
| US20140274794A1 (en) | Methods and Compositions for Diagnosis of Ovarian Cancer | |
| KR101219519B1 (en) | A method for the diagnosis using lectin | |
| CN108181457B (en) | Methods and kits for identifying, assessing, preventing and treating lung diseases | |
| EP2201363A2 (en) | Detection of saliva proteins modulated secondary to ductal carcinoma in situ of the breast | |
| EP4045912A1 (en) | Apparatuses and methods for detection of pancreatic cancer | |
| CN111735949B (en) | Wnt7a and CA125 combined as early ovarian cancer biomarker and kit | |
| JP4593117B2 (en) | Use of carbamoyl phosphate synthetase 1 (CPS1) and fragments thereof for diagnosing inflammatory diseases and sepsis | |
| TWI408370B (en) | A serological maker for detecting pancreatic cancer and a method for using the serological maker | |
| CN111735950B (en) | FGF18 and CA125 combined used as early ovarian cancer biomarker and kit | |
| JP4515099B2 (en) | Methods for diagnosing inflammatory diseases and infections by determining LASP-1 immunoreactivity | |
| Sánchez-Carbayo et al. | Urinary tissue polypeptide-specific antigen for the diagnosis of bladder cancer | |
| CN111912987B (en) | FGF18 and HE4 combined used as early ovarian cancer biomarker and kit | |
| CN116121392A (en) | Methods and reagents for diagnosis of pancreatic cystic tumours | |
| KR101219516B1 (en) | Polypeptide markers for the diagnosis of cancers and methods for the diagnosis of cancers using the same | |
| CN111781364B (en) | Wnt7a and HE4 combined as early ovarian cancer biomarker and kit | |
| CN111693703B (en) | Spondin1 and HE4 combined as early ovarian cancer biomarker and kit | |
| US20150004633A1 (en) | Assays and methods for the diagnosis of ovarian cancer | |
| CA2525740A1 (en) | Biomarkers for the differential diagnosis of pancreatitis and pancreatic cancer | |
| WO2010017331A1 (en) | Signal pathway alterations and drug target elevations in primary metachronous metastatic colorectal cancer compared to non-metastatic disease | |
| WO2023230584A1 (en) | Methods and systems for risk stratification and management of bladder cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |